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Phagocytosis Assay for α-Synuclein Fibril Uptake by Mouse Primary Microglia

小鼠初级小胶质细胞吸收α-突触核蛋白原纤维的吞噬分析

CD Cuilian Du
FZ Feifan Zhang
Claire Xi Zhang Claire Xi Zhang

发布: 2018年09月05日第8卷第17期 DOI: 10.21769/BioProtoc.2986 浏览次数: 6615

评审: Xi FengLalitha SrinivasanRosa Barreira da Silva

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Abstract

Microglia are professional phagocytes in the brain and deficiency in their phagocytic activity plays an important role in Parkinson’s disease. This protocol mainly describes the phagocytosis assay for uptake of α-synuclein preformed fibrils, a pathologic form of α-synuclein, by primary microglia.

Keywords: Phagocytosis (吞噬作用) search α-synuclein preformed fibrils (α-突触核蛋白原纤维) search Microglia (小胶质细胞) search Parkinson's disease (帕金森病) search Immunofluorescence (免疫荧光) search

Background

As the immune cells of the brain, microglia play critical roles in the central nervous system. In the physiological state, microglia constantly explore the surrounding environment and are involved in synaptic pruning. Microglia can be activated by any type of pathologic event or change in brain homeostasis (Wolf et al., 2017). Upon activation, microglia undergo morphological changes, proliferate, secrete inflammatory cytokines, migrate to the lesion site, and phagocytose pathogen, sick cells, the debris, even extracellular protein aggregates (Kettenmann et al., 2011; Fu et al., 2014). α-Synuclein is an abundant protein in neurons and is the principal component of the intraneuronal inclusions known as Lewy bodies and Lewy neurites in Parkinson’s disease (Luk et al., 2012). Recent studies showed that α-synuclein undergoes cell-to-cell spreading and microglia are the primary scavengers of α-synuclein, which likely take the burden of α-synuclein from neurons (Wolf et al., 2017). Here, we describe a protocol using human α-synuclein monomer to generate pre-formed fibrils and measuring the uptake of α-synuclein preformed fibrils by microglia phagocytosis (Du et al., 2017).

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-300 )
  2. Cover Glass (VWR, catalog number: 631-0150 )
  3. 50 ml centrifuge tube (Corning, catalog number: 430828 )
  4. 15 ml centrifuge tube (Corning, catalog number: 430790 )
  5. 75 cm2 flask (Corning, catalog number: 430641U )
  6. 24-well plate (Corning, Costar®, catalog number: 3524 )
  7. 40 μm cell strainer (Corning, Falcon®, catalog number: 352340 )
  8. 10 ml serological pipets (Corning, catalog number: 4488 )
  9. 3 kD centrifugal filter units (Merck, catalog number: UFC500324 )
  10. 30 kD centrifugal filter units (Merck, catalog number: UFC503024 )
  11. Column assembled with filter (in GST-tag Protein Purification Kit) (Beyotime Biotechnology, catalog number: P2262 )
  12. 0.22 μm filter (Merck, Millex, catalog number: SLGP033RB )
  13. Ampicillin (Sigma-Aldrich, catalog number: BP021 )
  14. Escherichia coli strain BL21 (DE3) (100 μl in 1.5 ml cryogenic vial, TIANGEN Biotech, catalog number: CB105 )
  15. pGEX-4T-2-GST (Obio technology)
  16. 20 C57BL/6 postnatal day 1 mice (Charles River Laboratories) without gender preferences
  17. Dulbecco's modified Eagle’s medium-F12 (GE Healthcare, HycloneTM, catalog number: SH30023.01 )
  18. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
  19. Isopropyl-β-D-thiogalactoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
  20. EDTA (Sigma-Aldrich, catalog number: E6758 )
  21. Lysozyme (Sigma-Aldrich, catalog number: 62971 )
  22. DNase1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18068015 )
  23. PMSF (Sigma-Aldrich, catalog number: P7626 )
  24. Protease inhibitor (Roche Diagnostics, catalog number: 04693132001 )
  25. Glutathione SepharoseTM 4B (GE Healthcare, catalog number: 17043001 )
  26. Bicinchoninic acid assay (BCA) kit (Thermo Fisher Scientific, catalog number: 23225 )
  27. Penicillin Streptomycin 100x Solution (GE Healthcare, HycloneTM, catalog number: SV30010 )
  28. Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1399 )
  29. Cytochalasin D (Abcam, catalog number: ab143484 )
  30. Saponin (Sigma-Aldrich, catalog number: 47036 )
  31. Anti-myc antibody (Santa Cruz Biotechnology, catalog number: SC789 )
  32. Alexa Fluor594 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific, catalog number: A-11007 )
  33. 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, catalog number: H-1200 )
  34. PBS (ZSGB-BIO, catalog number: ZLI-9062 )
  35. Yeast extract (Oxoid, catalog number: LP0021B )
  36. NaCl (Sigma-Aldrich, catalog number: V900058 )
  37. Tryptone (Oxoid, catalog number: LP0042B )
  38. Agarose (Biowest, catalog number: 111860 )
  39. Thrombin (Sigma-Aldrich, catalog number: T4648 )
  40. Tris-HCl (Sigma-Aldrich, catalog number: T1503 )
  41. KCl (MODERN ORIENTAL FINE CHEMISTRY, catalog number: JC-AR20140011 )
  42. Na2HPO4 (Beijing Chemical Factory, catalog number: A1060056 )
  43. KH2PO4 (Beijing Chemical Factory, catalog number: A1049020 )
  44. SDS (GE Healthcare, catalog number: 17131301 )
  45. 4% paraformaldehyde (ALADDIN, catalog number: C104188 )
  46. TritonTM X-100 (Sigma-Aldrich, catalog number: V900502 )
  47. Bovine serum albumin (Sigma-Aldrich, catalog number: B2064 )
  48. Nail polish (USHINE)
  49. LB medium (selection plate) (see Recipes)
  50. LB medium (1 L) (see Recipes)
  51. 0.01 M PBS (pH 7.4, 1 L) (see Recipes)
  52. Buffer A (pH 7.5) (see Recipes)

Equipment

  1. Pipettes (Eppendorf, model: Research® plus, catalog number: 3120000062 )
  2. Surgical instruments: ophthalmic scissors, curved ophthalmic forcep
  3. Dry bath (VIVO, catalog number: VHD150S )
  4. Cell culture CO2 incubator (Thermo Fisher Scientific, catalog number: 3131 )
  5. Shaker (Shanghai Meditry Instrument, catalog number: THZ-C-1 )
  6. Centrifuge (Eppendorf, model: 5430 R )
  7. Rotator (Cole-Parmer, Stuart, catalog number: SB3 )
  8. 4 °C RevcoTM High-Performance Laboratory Refrigerator (Thermo Fisher Scientific, catalog number: REC3004V )
  9. Stereomicroscope (ZEISS, model: Axio Vert.A1 )
  10. Ultrasonic processor (Cole-Parmer, catalog number: CN-04714-50 )
  11. Confocal microscopy (Leica Microsystems, model: Leica TCS SP8 )
  12. Biological safety cabinet (Thermo Fisher Scientific, catalog number: 51026654 )
  13. Eppendorf BioPhotometer (Eppendorf, catalog number: 6133000044 )

Software

  1. NIH ImageJ (http://fiji.sc/)
  2. SPSS software (SPSS 16.0, Chicago, IL)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Du, C., Zhang, F. and Zhang, C. X. (2018). Phagocytosis Assay for α-Synuclein Fibril Uptake by Mouse Primary Microglia. Bio-protocol 8(17): e2986. DOI: 10.21769/BioProtoc.2986.

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分类

神经科学 > 细胞机理 > 细胞分离和培养

免疫学 > 免疫细胞功能 > 巨噬细胞

细胞生物学 > 基于细胞的分析方法 > 内吞作用

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